Arylboronic acids, such as phenylboronic acids, are known to interact with a wide range of polar molecules having certain requisite functionalities. Complexes of varying stability, involving 1,2-diols, 1,3-diols, 1,2-hydroxy acids, 1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketones and 1,3-diketones, are known to occur with either neutral phenylboronic acid or phenylboronate anion. Immobilized phenylboronic acids have been used as chromatographic supports to selectively retain, from diverse biological samples, those molecular species having the requisite functionalities. Many important biological molecules including, but not limited to, carbohydrates, catecholamines, prostaglandins, ribonucleosides, and steroids contain the requisite functionalities, and have been either analyzed or purified in this manner. The use of phenylboronic acid chromatographic media for the isolation and separation of biological molecules has been discussed in several reviews (see, Singhal, R. P. and DeSilva, S. S. M. (1992) Adv. Chromatog., 31, 293-335; Mazzeo, J. R. and Krull, I. S. (1989) BioChromatog., 4, 124-130; and Bergold, A. and Scouten, W. H. (1983) in Solid Phase Biochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley & Sons, New York).
Molecular species having cis or coaxial 1,2-diol and 1,3-diol functionalities, and particularly carbohydrates, are known to complex with immobilized compounds having a phenylboronate anion to form cyclic esters under alkaline aqueous conditions (see, Lorand, J. P. and Edwards, J. O. (1959) J. Org. Chem., 24, 769). In addition, carbohydrate binding to arylboronic acid moieties is known to induce fluorescence changes by enhancing the electronic interaction between an arylboronic acid and an amine functionality contained within the arylboronic acid (see, T. D. James et al. (1994) J. Chem. Soc. Chem. Comm., 477-478).
In view of their ability to complex to biomolecules, such as biological macromolecules, reagents derived from arylboronic acids are useful in a variety of bioconjugation applications involving immobilization, purification and detection. Bioconjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule. Bioconjugation includes, but is not limited to, the conjugation of proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes and cells with each other or with any other molecular species that add useful properties. Immobilization of biological macromolecules is also considered a special case of bioconjugation, in which the macromolecule is conjugated, either reversibly or irreversibly, to an insoluble support. Bioconjugation is utilized extensively in biochemical, immunochemical and molecular biological research. Major applications of bioconjugation include, but are not limited to, detection of gene probes, enzyme-linked immuno solid-phase assay (ELISA), monoclonal antibody drug targeting and medical imaging.
In most instances, bioconjugation is based upon known reactions between two binding partners making a binding pair. One example of a bioconjugation reaction involves a first binding partner, e.g., an ortho-substituted acetamidophenylboronic acid, and a second binding partner, e.g., the vicinal diol moieties of the carbohydrate residues associated with glycoproteins (see, Cai, S. X. and Keana, J. F. W. (1991) Bioconjugate Chem., 2, 317-322).
In addition, phenylboronic acid bioconjugates derived from 3-isothiocyanatophenylboronic acid have been successfully utilized for appending radioactive technetium dioxime complexes to monoclonal antibodies for use in medical imaging (see, Linder, K. E., Wen, M. D., Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170; Linder, K. E., Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F., Narra, R. K. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 407-414).
Moreover, boronic acid reagents have exhibited broad utility as bioconjugation reagents when utilized in conjunction with newly developed boronic acid complexing reagents derived from salicylhydroxamic acid (SHA) and 2,6-dihydroxybenzohydroxamic acid (DHBHA). Boronic acid reagents, boronic acid complexing reagents, their conjugates and bioconjugates, as well as methods for their preparation and use are disclosed in U.S. Pat. Nos. 5,594,111, 5,623,055, 5,668,258, 5,648,470, 5,594,151, 5,668,257, 5,677,431, 5,688,928, 5,744,627, 5,777,148, 5,831,045 and 5,831,046.
In view of the usefulness of arylboronic acids in bioconjugation reactions, what is needed in the art are boronic acid compounds amenable to incorporation into synthetic oligonucleotides, such as during automated solid phase synthesis. The boronic acid-modified oligonucleotides thus produced would be useful in bioconjugation reactions, such as the immobilization, purification and detection of macromolecules. The present invention fulfills these and other needs.